Aspergillus spp. are seed-deteriorating fungi known for their ability to produce mycotoxins in crops such as maize, peanuts, tree nuts, almonds, Brazil nuts, pistachios, melon, pumpkin, sunflower seeds and cottonseed. Typically the mycotoxins produced by Aspergillus spp. during colonization are aflatoxin and sterigmatocystin. Aflatoxin is normally, produced by A. flavus and A. parasiticus. Sterigmatocystin is produced by several Aspergillus spp. including A. nidulans and A. versicolor.
Aflatoxins have several structural forms. For instance, Aspergillus flavus produces aflatoxin B.sub.1 and aflatoxin B.sub.2. Aspergillus parasiticus--the second of the two aflatoxin-producing fungi--produces, on the other hand, aflatoxin G.sub.1 and aflatoxin G.sub.2 as well as B.sub.1 and B.sub.2. Of these, aflatoxin B.sub.1 is the most toxic and carcinogenic. (Sterigmatocystin is similar in toxicity and carcinogenicity to aflatoxin B.sub.1.) It further is the most prevalent of the aflatoxins in contaminated feedstocks.
Aflatoxin and sterigmatocystin, end products of the same biosynthetic pathway, are both derived from polyketides which are bioreactive secondary metabolites that are synthesized like fatty acids. A lengthy biosynthetic pathway has been proposed: initial polyketide precursor.fwdarw.norsolorinic acid.fwdarw.averantin.fwdarw.averufanin.fwda rw.averufin.fwdarw.versiconal hemiacetal acetate.fwdarw.versicolorin B.fwdarw.versicolorin A.fwdarw.demethylsterigmatocystin.fwdarw.sterigmatocystin.fwdarw.O-methyls terigmatocystin.fwdarw.aflatoxin B.sub.1.
Infection of crops by Aspergillus spp. is highly undesirable since aflatoxin and the related mycotoxin, sterigmatocystin, are human carcinogens. In developing countries where governments cannot afford to screen and destroy contaminated food, high liver cancer rates are associated with aflatoxin/sterigmatocystin contamination.
In certain years, environmental conditions heavily favor the production of aflatoxin as well as sterigmatocystin. It is necessary to survey food products and feeds for such contamination. Contaminated supplies in the U.S. are typically destroyed though at times contaminated supplies can be treated. As might be expected, the surveillance and resulting expense associated with the sampling, decontaminating and/or destroying of food supplies represents a herculean task.
In an attempt to deal with the problem of aflatoxin/sterigmatocystin contamination of food supplies, regulatory agencies have imposed allowable limits. While European countries have typically imposed a 0 ppb limit, the United States currently has a 20 ppb limit for certain foods.
The ideal solution is to of course prevent the occurrence of aflatoxin/sterigmatocystin food and feed contamination. Traditional plant protection practices, such as through breeding, have been unsuccessful. Current studies have further included the placement of antifungal compounds into susceptible crops by genetic engineering techniques. Transformation of peanuts, cotton and walnuts with a variety of antifungal agents such as lytic peptides, bacterial endo-chitinase, tobacco osmotin, and chitinase have been made. Cary, J. W. et al., Construction of Transformation Vectors Expressing Resistance to A. flavus in Cotton, p. 16, Proceedings from the 7th Annual Aflatoxin Elim Workshop Meeting, St. Louis, Miss., J. Robens (ed.), USDA-ARS, Beltsville, Md. 20705, 1994; Chlan, C. et al., Transformation and Regeneration of Cotton to Yield Improved Resistance to A. flavus p. 15, Proceedings from the 7th Annual Aflatoxin Elim Workshop Meeting, St. Louis, Miss., J. Robens (ed.), USDA-ARS, Beltsville, Md. 20705, 1994; Dandekar, A. et al., Process in engineering walnuts for resistance to Aspergillus flavus, p. 18, Proceedings from the 7th Annual Aflatoxin Elim Workshop Meeting, St. Louis, Miss., J. Robens (ed.), USDA-ARS, Beltsville, Md. 20705, 1994; Li, Z. et al., Development of Gene Delivers Systems Capable of Introducing Aspergillus flavus--Resistance Genes into Peanuts p. 12, Proceedings from the 7th Annual Aflatoxin Elim Workshop Meeting, St. Louis, Miss., J. Robens (ed.), USDA-ARS, Beltsville, Md. 20705, 1994; Ozias-Akins, P. et al., Genetic Engineering of Peanut-Insertion of Four Genes that May Offer Disease Resistance Strategies, p. 14, Proceedings from the 7th Annual Aflatoxin Elim Workshop Meeting, St. Louis, Miss., J. Robens (ed.), USDA-ARS, Beltsville, Md. 20705, 1994; Weissinger, A. et al., Progress in the Development of Transgenic Peanut with Enhanced Resistance to Fungi, p. 13, Proceedings from the 7th Annual Aflatoxin Elim Workshop Meeting, St. Louis, Miss., J. Robens (ed.), USDA-ARS, Beltsville, Md. 20705, 1994. Unfortunately, no finding has yet been made that such antifungal agents function as a defense against the toxicity caused by Aspergillus spp.
As a second approach, attempts have been made to control aflatoxin/sterigmatocysin production through the use of biocontrol agents. For example, studies have focused on atoxigenic Aspergillus isolates to compete with toxigenic isolates. These have not yet succeeded in halting aflatoxin/sterigmatocystin contamination. Unsuccessful attempts have further been made on methods of promoting competitive microorganisms in the soil.
A third approach has been to identify those endogenous "anti-aflatoxin/Aspergillus" molecules in aflatoxin-free plants which have been demonstrated physiologically to function in vivo against Aspergillus spp. or which suppress the production of aflatoxin. A suggested model useful for the identification of such molecules is soybean (Glycine max L.),a leguminous species that produces oil rich seeds and which is virtually immune to Aspergillus-invasion and subsequent aflatoxin contamination. Howell, R. W., Mycotoxin in Research in Oil Seeds, pp. 61-66, Proc. 1st US-Japan Conference on Toxic Microorg., U.S. Department of Interior and UJNR Panels on Toxic Micro-Organisms, Washington, DC; Martinson, C. A. et al., Detecting Corn Germplasm with Resistance to Aflatoxin Production in Kernel Extracts, p. 8, Proceedings from the 7th Annual Aflatoxin Elim Workshop Meeting, St. Louis, Miss., J. Robens (ed.), USDA-ARS, Beltsville, Md. 20705, 1994. Further, it has been suggested that th- lipoxygenase pathway produces antifungal compounds that could be involved in keeping soybean free of aflatoxin contamination. Such compounds include the volatile C.sub.6 aldehydes hexanal or cis-3-hexenal (which isomerizes into trans-2-hexenal) from the 13-hydroperoxides of linoleic and linolenic acid by hydroperoxide lyases. See Doehlert D.C. et al., Evidence Implicating Lipoxygenase Pathway in Providing Resistance to Soybeans Against Aspergillus flavus 83:1473-1477, Phytopathology, 1993.
Other diversified studies have focused on the identification of a single gene product to transfer to susceptible crops which can inhibit Aspergillus growth and/or aflatoxin/sterigmatocystin biosynthesis. Some research endeavors have centered on transforming foreign genes into susceptible aflatoxin/sterigmatocystin crops. To date, such genes have not resulted in reducing or eliminating either aflatoxin or sterigmatocystin contamination.